Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation

Abstract

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.

Fig 1. The BOMB platform.

The BOMB platform is composed of (1) ferrite MNPs that can be coated with either a (2) silica or (3) carboxyl surface, BOMB magnetic racks produced by laser cutting or 3D printing, and (4–9) the basic BOMB protocols for purification of nucleic acids from various sample origins. The circled numbers indicate the protocols for the respective procedure. BOMB, Bio-On-Magnetic-Beads; MNP, magnetic nanoparticle; TNA, total nucleic acid.

Fig 2. Versatility of the BOMB protocols for nucleic acid isolation.

Nucleic acid extraction from various sample origins using the BOMB extraction protocols (S1 Appendix, BOMB protocols 4–8). (A) Size exclusion of GeneRuler DNA Ladder Mix (Thermo) using BOMB silica-coated magnetic beads (S1 Appendix, BOMB protocol 4.1). Different volumes of binding buffer compared to sample volume were used to achieve size exclusion. Two volumes of cBB were used as a control relative to input. (B) Gel extraction of CHD1 PCR products amplified from female chicken (Gallus gallus domesticus) gDNA. The two rightmost lanes contain the gel extracted bands from input lane using S1 Appendix, BOMB protocol 4.3 with carboxyl-coated magnetic beads. The volumes loaded are proportional (i.e., the right-hand 2 lanes represent the efficiency of capture from the left-hand lane). MW: Hyperladder IV (Bioline). (C) gDNA isolated from TOP10 Escherichia coli cells. The lanes labelled “gDNA” contain gDNA isolated from untransformed cells, whereas lanes denoted as “gDNA+plasmid” contain the total DNA extracted from E. coli carrying pHAGE-EFS plasmid (S1 Appendix, BOMB protocol 7.1). Purified pHAGE/EFS plasmid is shown in the “plasmid” lane (S1 Appendix, BOMB protocol 5.1). MW: GeneRuler DNA Ladder Mix (Thermo). (D) Total plasmid DNA yield (μg) extracted from E. coli, plotted against the A260 nm/A280 nm ratio for each sample. DNA concentration and purity were measured with UV-Spectroscopy (NanoDrop). Black dots represent samples extracted using the S1 Appendix, BOMB protocol 5.1; red dots represent samples processed using a commercial kit. (E) TNA isolation (S1 Appendix, BOMB protocol 6.6) from E. coli (left two lanes) followed by DNase I digest (right two lanes). MW: GeneRuler DNA Ladder Mix (Thermo). (F) Isolation of TNA from HEK293 cells using the S1 Appendix, BOMB protocol 6.1 (lanes 6 and 7) followed by digestion with either DNase I (lanes 2 and 3) or RNase A (lanes 4 and 5). (G) gDNA isolated from 500,000 HEK293 cells using S1 Appendix, BOMB protocol 7.1. (H) Total RNA isolated from 500,000 HEK293 cells following S1 Appendix, BOMB protocol 8.1. (I) Total DNA yield (μg) of representative extractions from HEK293 cells, plotted against the A260 nm/A280 nm ratio for each sample. (J) Total RNA yield (μg) of representative extractions from HEK293 cells, plotted against the A260 nm/A280 nm ratio for each sample. (K) qPCR amplification curve of a 10-fold serial dilution (black: undiluted; dark grey: 1:10 diluted; light grey: 1:100 diluted) of RNA from HEK293 cells reverse-transcribed into cDNA. (L) gDNA isolated from various rabbit (Oryctolagus cuniculus) tissues using BOMB silica-coated magnetic beads following S1 Appendix, BOMB protocol 6.3. MW: Hyperladder I (Bioline). (M) TNA isolation from yeast (Saccharomyces cerevisae) using S1 Appendix, BOMB protocol 6.5. MW: GeneRuler DNA Ladder Mix (Thermo). (N) TNA isolation from clover (Trifolium repens), daisy (Bellis perennis), and ryegrass (Lolium perenne) according to S1 Appendix, BOMB protocol 6.4 with carboxyl-coated magnetic beads. MW: Hyperladder I (Bioline). (O) Total DNA yield (ng) of representative extractions from leaves of T. repens, plotted against the A260 nm/A280 nm ratio of each sample. (P) TNA isolation from 50 ml of lake water following S1 Appendix, BOMB protocol 6.7. MW: GeneRuler DNA Ladder Mix (Thermo). Underlying data for Fig. 2 can be found in S1 Data. BOMB, Bio-On-Magnetic-Beads; cBB, commercial binding buffer; CHD1, Chromodomain Helicase DNA Binding Protein 1; eTNA, environmental total nucleic acid; gDNA, genomic DNA;qPCR, quantitative polymerase chain reaction; TNA, total nucleic acid.

Fig 3. The BOMB platform is both modular and universal.

Purification of nucleic acid from a wide variety of biological and synthetic input sources (left-hand panel) can be performed using the BOMB platform. Following initial extraction using BOMB beads (central bead icon), the resulting nucleic acids, DNA, RNA, or TNA, can be further manipulated and repurified using BOMB protocols and/or passed to final analysis methods or reintroduced back into a biological system. BOMB, Bio-On-Magnetic-Beads; NGS, next-generation sequencing; qPCR, quantitative polymerase chain reaction; TNA, total nucleic acid.